A multivalent approach to the discovery of long-acting β(2)-adrenoceptor agonists for the treatment of asthma and COPD

A multivalent approach was applied to the design of long-acting inhaled β(2)-adrenoceptor agonists. A series of dimeric arylethanolamines based on the short acting β(2)-adrenoceptor agonist albuterol were prepared, varying the nature and length of the linker between the basic nitrogens. None of the C(2)-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine, was found to have increased binding potency in vitro relative to the parent monomer. Optimization of this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and long duration of action in a guinea pig model of bronchoprotection.     

Discovery of substituted phenyl urea derivatives as novel long-acting β2-adrenoreceptor agonists

The synthesis of diverse functionalized ureas in a semi-parallel fashion is described, as well as their β₁/β₂-adrenergic activities and the corresponding structure-activity relationship (SAR). We have focused on lipophilicity and duration of action, and we have discovered a strong correlation in this series of molecules. A quantitative structure-activity relationship (QSAR) analysis will be presented that quantifies this relationship.     

From libraries to candidate: the discovery of new ultra long-acting dibasic β₂-adrenoceptor agonists

Libraries of dibasic compounds designed around the molecular scaffold of the DA(2)/β(2) dual agonist sibenadet (Viozan?) have yielded a number of promising starting points that have been further optimised into novel potent and selective target molecules with required pharmacokinetic properties. From a shortlist, 31 was discovered as a novel, high potency, and highly efficacious β(2)-agonist with high selectivity and a duration of action commensurable with once daily dosing.     

Synthesis and evaluation of two series of 4′-aza-carbocyclic nucleosides as adenosine A2A receptor agonists

The synthesis of two series of 4′-aza-carbocyclic nucleosides are described in which the 4′-substituent is either a reversed amide, relative to the carboxamide of NECA, or an N-bonded heterocycle. Using established purine substitution patterns, potent and selective examples of agonists of the human adenosine A_2A receptor have been identified from both series. The propionamides 14–18 and the 4-hydroxymethylpyrazole 32 were determined to be the most potent and selective examples from the 4′-reversed amide and 4′-N-bonded heterocyclic series, respectively.     

Dual β2-adrenoceptor agonists-PDE4 inhibitors for the treatment of asthma and COPD

We designed and synthesized a novel class of dual pharmacology bronchodilators targeting both β(2)-adrenoceptor and PDE4 by applying a multivalent approach. The most potent dual pharmacology molecule, compound 29, possessed good inhibitory activity on PDE4B2 (IC(50)=0.278 μM, which was more potent than phthalazinone, IC(50)=0.520 μM) and possessed excellent relaxant effects on tracheal rings precontracted by histamine (pEC(50)=9.3).     

Design-driven LO: the discovery of new ultra long acting dibasic β2-adrenoceptor agonists

Starting with the molecular scaffold of the DA₂/β₂ dual agonist sibenadet (Viozan™), a number of molecular changes were incorporated, which were designed to increase the potency and selectivity of the target molecule, and improve its pharmacokinetics. Through this process a novel, high potency, full β₂-agonist with high selectivity and long duration capable of being dosed once daily has been discovered.     

Crystal structures of a stabilized β1-adrenoceptor bound to the biased agonists bucindolol and carvedilol

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Highlights? Structures of β1AR bound to the biased agonists bucindololol and carvedilol ? The biased agonists form unique contacts with β1AR not seen with other antagonists ? The structures explain the pharmacological differences in the Arg389Gly polymorphism     

Discovery of olodaterol,a novel inhaled β2-adrenoceptor agonist with a 24 h bronchodilatory efficacy

Compound 4p was identified from a series of 6-hydroxy-4H-benzo[1,4]oxazin-3-ones as potent agonist of the human β₂-adrenoceptor with a high β₁/β₂-selectivity. A complete reversal of acetylcholine-induced bronchoconstriction which lasted over the whole study period of 5 h was demonstrated for 4p in a guinea pig in vivo model without any signs of cardiovascular effects up to 10-fold above the first dose reaching 100% bronchoprotection. The enantiomerically pure (R)-form of 4p exerted a bronchodilatory efficacy over 24 h in dogs and guinea pigs in the absence of systemic pharmacodynamic effects. Formoterol which was tested as comparator in the same in vivo models of acetylcholine-induced bronchoconstriction did not retain efficacy after 24 h. In summary, the preclinical profile of compound (R)-4p (olodaterol, also known as BI 1744 CL) suggests a potential for once-daily dosing in man accompanied with an improved safety profile.     

Design,synthesis and evaluation of dual pharmacology β2-adrenoceptor agonists and PDE4 inhibitors

A novel series of formoterol–phthalazinone hybrids were synthesised and evaluated as dual pharmacology β₂-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high β₂-adrenoceptor agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist (EC₅₀ = 1.05 nM, pEC₅₀ = 9.0) and potent PDE4B2 inhibitory activities (IC₅₀ = 0.092 μM).     

Evaluation of the Salivette as sampling device for monitoring β-adrenoceptor blocking drugs in saliva

The Salivette® was evaluated with a range of racemic β-adrenoceptor blocking drugs with different lipophilicity. Recovery from the Salivette appeared to be independent of the stereochemical configuration of the drugs but a significant loss of drug due to the Salivette was observed for all tested drugs. The performance of the method, in terms of accuracy and precision, fitted well within the generally accepted criteria for validation, except near the limit of quantification. The Salivette is successfully used for quantitating salivary β-blocking drugs.     

Investigation of piperazine benzamides as human β3 adrenergic receptor agonists for the treatment of overactive bladder

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human β₃-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established β₃ potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.     

Synthesis and structure–activity relationships of a series of 3-aryl-4-isoxazolecarboxamides as a new class of TGR5 agonists

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent agonists of the human TGR5 G-protein-coupled receptor is described. Many analogues were readily accessible via solution-phase synthesis which resulted in the rapid identification of key structure–activity relationships (SAR), and the discovery of potent exemplars (up to pEC₅₀ = 9). Details of the SAR and optimization of this series are presented herein.     

Effects of α2- and β-adrenoceptor agonists on growth hormone secretion following lesion of the noradrenergic system of the rat

The aim of the present investigation was to lesion the noradrenergic system and to measure the effect on growth hormone (GH) secretion following peripheral administration of ₂- and -adrenoceptor agonists. Direct injection of these agonists into the paraventricular nucleus of the hypothalamus (PVN) and its effect on GH secretion were also investigated. Systemic administration of N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP₄, 60 mg/kg, injected i.p. 10 days prior to experimentation) significantly decreased the noradrenaline (NA) content of the hippocampus, frontal cortex and hypothalamus but had no effect on the dopamine (DA) or serotonin (5-HT) content of these areas. Bilateral injection of 6-hydroxydopamine (6-OHDA, 10 g/l, 14 days prior to experimentation) into the medial forebrain bundle (MFB) caused a greater reduction of NA and also decreased the DA and 5-HT content of the hypothalamus. Analysis of the PVN of the hypothalami of rats following 6-OHDA lesion of the MFB showed significantly decreased NA and 5-HT content. Neither DSP₄ treatment nor 6-OHDA lesion of the MFB affected the clonidine (250 g/kg, i.p.) induced stimulation of GH secretion. Injection of isoproterenol (1 mg/kg, i.p.) had varying effects on GH secretion. It stimulated GH release in control rats but not in DSP₄ or MFB lesioned rats. Direct injection of clonidine (0.1 g/l) into the PVN significantly stimulated GH secretion, whereas injection of isoproterenol (2.5 g/l) into the PVN did not affect GH levels when compared to controls. The results of the present study do not support the hypothesis that hypoactivity of the central noradrenergic system may be the cause of the blunted GH response to clonidine observed in depressed patients.     

Structure-activity relationships in a series of C2-substituted gluco-configured tetrahydroimidazopyridines as β-glucosidase inhibitors

Inhibition of glycoside hydrolases has widespread application in treatment of diabetes, viral infections, lysosomal storage diseases and cancers. Gluco-configured tetrahydroimidazopyridines are the most potent β-glucosidase inhibitors reported to date. Using transition state mimic strategy, a series of C2-substituted gluco-configured tetrahydroimidazopyridines were designed and synthesized. Compounds 3 (K(i)=0.64 nM) and 5 (K(i)=0.58 nM) showed stronger inhibitory potency against β-glucosidase. Maestro 9.1 was used to study the structure-activity relationships by docking the compounds into the β-glucosidase active sites.     

Should inhaled anticholinergics be added to β2 agonists for treating acute childhood and adolescent asthma? A systematic review

Objectives To estimate the therapeutic and adverse effects of addition of inhaled anticholinergics to β₂ agonists in acute asthma in children and adolescents.Design Systematic review of randomised controlled trials of children and adolescents taking β₂ agonists for acute asthma with or without the addition of inhaled anticholinergics.Main outcome measures Hospital admission, pulmonary function tests, number of nebulised treatments, relapse, and adverse effects.Results Of 37 identified trials, 10 were relevant and six of these were of high quality. The addition of a single dose of anticholinergic to β₂ agonist did not reduce hospital admission (relative risk 0.93, 95% confidence interval 0.65 to 1.32). However, significant group differences in lung function supporting the combination treatment were observed 60 minutes (standardised mean difference −0.57, −0.93 to −0.21) and 120 minutes (−0.53, −0.90 to −0.17) after the dose of anticholinergic. In contrast, the addition of multiple doses of anticholinergics to β₂ agonists, mainly in children and adolescents with severe exacerbations, reduced the risk of hospital admission by 30% (relative risk 0.72, 0.53 to 0.99). Eleven (95% confidence interval 5 to 250) children would need to be treated to avoid one admission. A parallel improvement in lung function (standardised mean difference −0.66, −0.95 to −0.37) was noted 60 minutes after the last combined inhalation. In the single study where anticholinergics were systematically added to every β₂ agonist inhalation, irrespective of asthma severity, no group differences were observed for the few available outcomes. There was no increase in the amount of nausea, vomiting, or tremor in patients treated with anticholinergics.Conclusions Adding multiple doses of anticholinergics to β₂ agonists seems safe, improves lung function, and may avoid hospital admission in 1 of 11 such treated patients. Although multiple doses should be preferred to single doses of anticholinergics, the available evidence only supports their use in school aged children and adolescents with severe asthma exacerbation.

Key messages

• The addition of multiple doses of anticholinergics to β₂ agonist inhalations seems indicated in the initial management of children and adolescents with severe exacerbations of asthma (⩽55% of predicted FEV₁)
• For the larger group of children and adolescents with mild to moderate asthma exacerbations, there is no apparent benefit from adding a dose of anticholinergics to β₂ agonists
• Little evidence exists to support the systematic addition of anticholinergics to every β₂ agonist inhalation, irrespective of patients’ disease severity

     

Rat β-glucuronidase as a reporter protein for the analysis of the plant secretory pathway

Abstract

E. coliβ-glucuronidase, a cytosolic enzyme, was found not to be a good reporter enzyme for secretion studies in plants. In this study, we chose to test and adapt an animal β-glucuronidase as a better reporter protein for the secretory pathway of plants. We modified rat β-glucuronidase to obtain secreted and vacuolar variants. Five different C-termini were produced: the original C-terminus of the rat enzyme, a 19 codon deletion (Δ19), a 15 codon deletion (Δ15) and fusions of the Δ19 or Δ15 termini with the last 6 or 7 codons of the vacuolar sorting determinant of tobacco chitinase A, respectively. The signal sequence of the rat β-glucuronidase polypeptide was replaced by the sequence encoding the signal peptide of tobacco chitinase A. In a transient expression system, the best enzymatic activity was found with β-glucuronidase having the 15 codons deletion, therefore Δ15 (secRGUS) and Δ15 + Chi (RGUS-Chi) were further evaluated and their efficiency of secretion or vacuolar targeting were tested under different conditions. To determine the correct targeting of reporter genes, we compared the localization of β-glucuronidase and of an endogenous marker, α-mannosidase. Treating cells with drugs that specifically affect different aspects of the secretory pathway also tested the validity of RGUS-based reporters. A non-specific inhibitor such as cytochalasin D and a wide range inhibitor such as BFA were compared with specific inhibitors such as wortmannin and bafilomycin A1. Finally, monensin and NH₄Cl were used to evaluate the role of vacuolar pH in correct RGUS-Chi targeting. The two new reporter proteins proved to be good tools for our studies in the transient expression system in tobacco protoplasts and for further applications.     

Hybrids consisting of the pharmacophores of salmeterol and roflumilast or phthalazinone: Dual β2-adrenoceptor agonists-PDE4 inhibitors for the treatment of COPD

A novel class of dual pharmacology bronchodilators targeting both β₂-adrenoceptor and PDE4 was designed and synthesised by combining the pharmacophores of salmeterol and roflumilast or phthalazinone. All the compounds exhibited better β₂-adrenoceptor agonist activities (pEC₅₀ = 8.47–9.20) than the reference compound salmeterol (pEC₅₀ = 8.3) and good inhibitory activity on PDE4B2 (IC₅₀ = 0.235–1.093 μM).     

Synthesis and structure–activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists

A series of fused-pyrimidine derivatives have been discovered as potent and orally active GPR119 agonists. A combination of the fused-pyrimidine structure and 4-chloro-2,5-difluorophenyl group provided the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivative 14a as a highly potent GPR119 agonist. Further optimization of the amino group at the 4-position in the pyrimidine ring led to the identification of 2-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetamide (16b) as an advanced analog. Compound 16b was found to have extremely potent agonistic activity and improved glucose tolerance at 0.1 mg/kg po in mice. We consider compound 16b and its analogs to have clear utility in exploring the practicality of GPR119 agonists as potential therapeutic agents for the treatment of type 2 diabetes mellitus.     

Discovery of novel α1-adrenoceptor ligands based on the antipsychotic sertindole suitable for labeling as PET ligands

The synthesis and in vitro preclinical profile of a series of 5-heteroaryl substituted analogs of the antipsychotic drug sertindole are presented. Compounds 1-(4-fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-(pyrimidin-5-yl)-1H-indole (Lu AA27122, 3i) and 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-(1-methylpiperidin-4-yl)-1H-indole (3l) were identified as high affinity α_1A-adrenoceptor ligands with K_i values of 0.52 and 0.16 nM, respectively, and with a >100-fold selectivity versus dopamine D₂ receptors. Compound 3i showed almost equal affinity for α_1B- (K_i = 1.9 nM) and α_1D-adrenoceptors (K_i = 2.5 nM) as for α_1A, as well as moderate affinity for 5-HT_1B (K_i = 13 nM) and 5-HT₆ (K_i = 16 nM) receptors, whereas 3l showed >40-fold selectivity toward all other targets tested. Based on in vitro assays for assessment of permeability rates and extent, it is predicted that both compounds enter the brain of rats, non-human primates, as well as humans, and as such are good candidates to be carried forward for further evaluation as positron emission tomography (PET) ligands.     

Behaviour of the NO-β-d-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for β-glucuronidase

1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-beta-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary beta-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1-->4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for beta-glucuronidase. 2. Mammalian and bacterial beta-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein beta-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4.1 and the Michaelis constant, K(m), is 3.3x10(-4)m with purified mouse urinary beta-glucuronidase as the enzyme source acting on the NO-beta-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mmu). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-beta-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.